The objective of this proposal is to study the biochemistry and genetics of lipoamide dehydrogenase of Pseudomonas putida. The basis of a new study of this enzyme is that we have recently discovered a specific lipoamide dehydrogenase for branched chain keto acid dehydrogeanse in P. putida which puts a new perspective on the structure and function of keto acid dehydrogenases. Pseudomonas putida grown on valine forms two lipoamide dehydrogenases with molecular weights of 56k and 49k. The 49k protein, lpd-val, functions with branched chain keto acid dehydrogenase but not with a partially purified preparation of 2-ketoglutarate dehydrogenase. The 56k protein, lpd-glu, is the only lipoamide dehydrogenase found in P. putida grown on glucose and functions with 2-ketoglutarate but not with branched chain keto acid dehydrogeanse. Escherichia coli forms only one lipoamide dehydrogenase for both pyruvate and 2-ketoglutarate dehydrogenases. Mammals form several isozymes of lipoamide dehydrogenase, but they appear to be conformational isomers of the same protein. These findings have potential significance for our understanding of genetic diseases such as maple syrup urine disease and Friedreich's ataxia and for those metabolic pathwyas such as glycine oxidation which also involve lipodamide dehydrogenase. The specific research plan include (1) purification of pyruvate of 2-ketoglutarate dehydrogenases from P. putida, (2) isolation and characterization of mutants of P. putida deficient in lipoamide dehydrogenase (3) determination of the amino acid composition and peptide maps of both lpd-glu and lpd-val and (4) if the genetic data indicate a single lpd structural gene, then we will search for an enzyme which converts lpd-glu to lpd-val.